ABSTRACT Contrast-enhanced ultrasound (CEUS) is the ideal imaging modality to investigate kidney disease. Ultrasound contrast agents are safe for diseased kidneys because the contrast agent is confined to the vascular space due to the agent?s size. This vascular confinement allows for the noninvasive quantification of blood flow and perfusion in the kidney. During initial studies to characterize diabetic kidney disease (DKD) with CEUS in a streptozotocin (STZ) model, atypical contrast patterns were found in diabetic rat kidneys using a novel image processing technique, called cumulative contrast images (CCI). In CCI, the time-dependent contrast refill of a flash-replenishment CEUS sequence is displayed in a single image. In the CCI of the diabetic kidneys, there was a pronounced reduction in contrast signal in the area where the cortex and medulla meet, or the corticomedullary region. This reduction in contrast is either caused by an anatomical change such as a reduction in the number of blood vessels or a functional adaptation such as vasoconstriction. The objective of this study is to determine the underlying condition that causes this reduction in contrast signal in the CCIs of diabetic kidneys. The first specific aim will optimize a new ultrafast CEUS imaging protocol that will allow for more accurate measurement of perfusion. The second specific aim will investigate the anatomical changes and functional alterations that may cause the loss of contrast in CCI by using analysis of glomerular filtration rate, biomarkers from blood samples, arteriograms of the kidney vasculature and pathological examination of tissues. Data collected from each specific aim will be compared to CCI contrast enhancement signal and kidney blood flow metrics computed from CEUS flash-replenishment imaging acquired longitudinally during early diabetes progression in a STZ model of DKD. This small project from Dr. Johnson, a new investigator, will be appropriate for the R03 model as it will achieve the stated preclinical studies in two years, providing crucial preliminary data to expand the project in the future. The outcome of this project will push forward the technology of CEUS imaging methods and is the next step in the effort to translate CEUS as an early biomarker of progressive DKD from preclinical tool to clinical application.